Use of amphotericin B derivatives as protease inhibitors

ABSTRACT

The invention is directed toward the use of amphotericin B and derivatives thereof as inhibitors of serine-proteases and for applying these inhibitors for the production of medicinal products intended for the treatment of infection by the human immunodeficiency virus (HIV).

The present invention relates to the use of derivatives of polyenemacrolides, in particular amphotericin B, as protease inhibitors, and tothe applications of said inhibitors for the production of medicinalproducts intended in particular for the treatment of infection by thehuman immunodeficiency virus (HIV).

Since the identification of HIV-1 and HIV-2 as agents for AIDS, numerousresearch studies have been carried out with the aim of understanding themechanism of viral infection and of obtaining effective antiviraltreatments.

Until now, nucleoside analogues such as AZT(3'-azido-2',3'-dideoxythymidine) and ddI (2',3'-dideoxyinosine) havebeen used for the treatment of AIDS and have resulted in an improvementof the clinical condition of patients. Unfortunately, severe sideeffects of treatment with AZT, in particular a degeneration of bonemarrow, have been reported, and moreover, many patients do not toleratethe drug very well or they become insensitive to its beneficial effects.

In the case of ddI, its use is still only at the experimental stage andits efficacy requires further investigation.

Furthermore, it has been shown that amphotericin B (AmB) and some of itsderivatives are active in vitro against several DNA and RNA virusescontaining a lipid envelope, and against various herpes virus strains.The methyl ester of amphotericin B (AME) SCHAFFNER et al., Biochem.Pharm., 35, 4110-4113, 1986! as well as liposome-encapsulatedamphotericin B PONTANI et al., Antiviral. Res. 11, 119-126 (1989)! havealso been described as inhibitors of cell death and of viral expressionin T cells infected in vitro with HIV-1. Moreover, since it is knownthat amphotericin B and its derivatives interact with cellular membranecholesterol, which causes fluidification of said membranes, it wasassumed that this mechanism was responsible for their antiviralactivity, either by destroying the virus or by preventing itspenetration into the target cells. HANSEN et al. Antiviral Research, 14,149-160, (1990)! have studied the in vitro activity of variousamphotericin derivatives on the HIV-1 virus. According to the results ofthis study, AME acts as a cell protecting agent by inhibiting theinfection of cells by free HIV: incubation of healthy lymphocytes in thepresence of AME prior to infection in vitro with an HIV isolate inhibitsthe infection; on the other hand, when HIV-infected lymphocytes arepreincubated in the presence of AME, their capacity to fuse withuninfected cells so as to form syncytia is increased. Another of thederivatives studied, MCG {NN'-(2,4'-methylmorpholino)-N"-ethylguanyl!amphotericin B}, does notpossess, according to this study, cell-protecting properties, but has adirect antiviral action: in the case of MCG, it is the preincubationwith the virus and not with the cells which inhibits vital infection.

Amphotericin B is currently used in patients suffering from AIDS, in thetreatment of infections caused by opportunistic microbes, in particularof deep-rooted mycoses which frequently occur in these patients.Clinical studies have shown that an antifungal therapy has to bemaintained indefinitely in order to avoid relapses, or even used as apreventive measure. However, frequent side effects such as fever orserious nephrotoxicity are associated with prolonged treatment usingamphotericin B.

By seeking out more active antifungal agents having a reduced toxicityand an enhanced efficacy, the inventors recently obtained, bysubstituting the mycosamine functional group of amphotericin B by a1-amino-1-deoxyketose which can itself be substituted, a series of newderivatives which are more soluble and less toxic than amphotericin B;these derivatives and their use as antifungal agents are the subject ofEuropean Patent Application No. 428 440.

In fact, the inventors have now tested the action of some of thesederivatives of formula (I) below ##STR1## in which: R1 represents themacrocyclic portion of the polyene macrolide,

R2 represents a hydrogen atom or a methyl group, and

R3 represents a methyl, ethyl, propyl or phenyl group,

as inhibitors of HIV infection.

They have thus observed that these derivatives inhibit thecytopathogenic effects induced by HIV-1, HIV-2and SIV (simianimmunodeficiency virus) and almost completely inhibit the replication ofHIV in all the cell types tested, at concentrations substantially belowthe cytotoxic concentrations.

Experiments carried out by the inventors in order to elucidate themechanism of action of the derivatives of general formula (I) have shownthat they are inactive on reverse transcriptase in vitro, and active onAZT-resistant HIV mutants.

Moreover, the anti-HIV activity of these derivatives exhibits unexpectedcharacteristics in view of the mechanisms of action described in theprior art for other amphotericin B derivatives.

In particular, the inventors have observed that, in contrast to what hasbeen described in the case of AME (HANSEN et al., publication mentionedabove), neither the pretreatment of cells with the AmB derivatives offormula (I) before infection, nor the pretreatment of the virus itself,have an inhibitory effect on viral replication.

Moreover, the inventors have observed that the derivatives of formula(I) inhibit the cytopathological effects of the human rhinitis virus(HRV14) in cultures of Hela-Ohio cells; yet, unlike the HIV-1 virus, theHRV14 virus is not an enveloped virus.

The inventors have therefore made the hypothesis that a commonmechanism, different from the interaction with the cell membranecholesterol and/or the viral envelope, may be involved in the anti-HIVand anti-Rhinovirus activities of the derivatives of general formula(I).

Moreover, the inventors have observed that the derivatives of formula(I) constitute protease inhibitors.

Now, the multiplication of HIV-1, like that of the Rhinovirus, requiresthe action of proteolytic enzymes: in the case of the Rhinovirus, twoproteases participate in the maturation of the polyprotein derived fromthe translation of the viral RNA; in the case of HIV-1, at least onecellular protease may play a role in the fusion between the viralenvelope and the cellular membrane, as well as in the formation ofsyncytia, and a viral protease (protease p10) may participate in thematuration of viral proteins.

The derivatives of formula (I) are inactive in vitro on protease p10,which is an aspartyl protease; in contrast, they are active on trypsinand on other serine proteases.

The inventors have investigated into whether other amphotericin Bderivatives possess the same protease-inhibiting activity; they observedthat derivatives in which the mycosamine functional group is substitutedby a 1-amino-1-deoxyketose group, such as those described in EuropeanApplication No. 428 440, have an activity similar to that of thederivatives of formula (I); moreover, amphotericin B, as well as all itsderivatives which have been tested by the inventors, possess, althoughto very different degrees, a certain inhibitory activity on trypsin; theextent of this activity seems to be linked to the presence of asubstituent on the mycosamine.

The new properties of the amphotericin B derivatives demonstrated by theinventors, and in particular the serine protease-inhibiting properties,make it possible to propose new uses for these derivatives. Inparticular, those of these derivatives with substantial serineprotease-inhibiting activity can be used in the treatment of diseasesinvolving a pathogenic agent whose multiplication cycle requires atleast one proteolytic enzyme of the serine protease type.

The subject of the present invention is the use of amphotericin B, or ofa derivative thereof, for the production of a protease inhibitor whichis active in particular on serine proteases such as trypsin, kallicreinand leucocyte elastase.

According to a preferred embodiment of the present invention, theamphotericin B derivative used is chosen from the derivatives whosetrypsin-inhibiting activity is at least twice greater than that ofamphotericin B.

According to another preferred embodiment of the present invention, themycosamine functional group of the amphotericin B derivative used issubstituted.

According to a preferred arrangement of this embodiment, the mycosaminefunctional group is substituted by an aspartyl group.

According to another preferred arrangement of this embodiment, themycosamine functional group is substituted by a 1-amino-1-deoxyketosegroup which is itself substituted where appropriate.

According to a preferred form of this arrangement, the derivative usedis of the formula (I), ##STR2## in which: R1 represents the macrocyclicportion of a polyene macrolide, preferably amphotericin B,

R2 represents a hydrogen atom or a methyl group, and

R3 represents a methyl, ethyl, propyl or phenyl group.

The subject of the present invention is also the use of the serineprotease inhibitors as defined above for the production of products, inparticular medicinal products, which inhibit the multiplication of apathogenic agent whose multiplication cycle requires the action of atleast one proteolytic enzyme of the serine protease type.

The amphotericin B derivatives which are preferred for this use arethose whose trypsin-inhibiting activity is at least twice greater thanthat of amphotericin B.

According to a preferred embodiment of the present invention, the saidpathogenic agent is a virus.

According to a preferred arrangement of this embodiment, said virus isthe HIV-1 virus.

According to another preferred arrangement of this embodiment, saidvirus is the HIV-2 virus.

According to another preferred arrangement of this embodiment, saidvirus is the SIV virus.

According to yet another preferred arrangement of this embodiment, saidvirus is a Rhinovirus, in particular the human Rhinitis virus.

According to another preferred embodiment of the present invention, saidpathogenic agent is a bacterium.

According to another preferred embodiment of the present invention, saidpathogenic agent is a eucaryotic organism, a parasite for man or foranimals.

The present invention will be more clearly understood by means of theadditional description below which refers to detailed examples relatingto the evaluation of the protease-inhibiting effect of variousamphotericin B derivatives, and of the activity on HIV, SIV and HRV14 ofa trypsin-inhibiting derivative,1-deoxy-1-amino-4,6-O-benzylidene-D-fructosyl-AmB (termed belowDABF-AmB).

However, it is evident that these examples are given solely by way ofillustration of the subject of the invention and in no manner constitutea limitation thereof.

I) DETERMINATION OF THE INHIBITORY ACTIVITY OF VARIOUS AMPHOTERICINDERIVATIVES ON SERINE PROTEASES 1) Inhibition of Trypsin

The different derivatives tested (AmB, AmB-methyl ester (AME),N-acetyl-AmB, N-aspartyl-AmB, N-acetyl-AME, DABF-AmB) were added, at aconcentration of 1 μM, to a 3.6×10⁻⁹ M solution of trypsin in a 0.1MTris-HCl buffer, pH 8.2, in the presence of 0.35 mM of DTNB; thereaction is initiated by adding the amount of substrate(Z-lysinthiobenzyl ester) required for a final concentration of 0.6×10⁻⁶M. The reaction is monitored by measuring the optical density at 412 nm.

The results are represented in FIG. 1; they are expressed in %inhibition relative to a control without inhibitor. Under the conditionsof the experiment, DABF-AmB inhibits the activity of trypsin by morethan 60%; the other derivatives tested have some activity (from about10% inhibition for AmB and AME up to about 40% inhibition forN-aspartyl-AmB), but none possesses an activity comparable to that ofDABF-AmB. Moreover, it was shown that benzylidene glucoside alone has noinhibitory action.

Table I shows the inhibition effect of increasing concentrations ofDABF-AmB on trypsin. Total inhibition is observed for a concentration of10⁻⁴ M.

                  TABLE 1    ______________________________________    % OF INHIBITION OF TRYPSIN BY DABF-AmB    AND AMB           10.sup.-7 M                   10.sup.-6 M                             10.sup.-5 M                                       10.sup.-4 M    ______________________________________    DABF-AmB 20        50        95      100    AMB       5        10        30      NT*    ______________________________________     *No tested.

These results suggest that the functional group involved in theprotease-inhibiting effect is present in the AmB molecule but that it isnot accessible as long as the modification of solubility and behavior insolution has not been achieved. The presence of a substituent on the NH₂group of mycosamine appears to increase the inhibitory activity of themolecule.

2) Activity of DABF-AmB on Various Proteases

DABF-AmB was tested, at a concentration of 1 μM, on various proteases(papain, chymotrypsin, kallicrein, leucocyte elastase, cathopsin D andrecombinant protease p10) under conditions and in the presence ofsubstrates appropriate for each of the proteases tested.

The results are represented in FIG. 2, in percentage of inhibitionobserved for trypsin under the same conditions. DABF-AmB inhibitskallicrein (results not shown) and leucocyte elastase which, liketrypsin, are serine proteases, but in contrast does not inhibitchymotrypsin. DABF-AmB does not inhibit papain (cysteine protease) norcathopsin D and protease p10 (aspartyl proteases).

An essential component of the activity of DABF-AmB on HIV and SIVviruses is certainly due to its action on the serine proteases of thecellular membrane, which play a part in vital infection.

The measurement of the inhibitory activity of amphotericin B derivativeson a serine protease such as trypsin can constitute a screening methodallowing the selection of the derivatives which are most active on HIV.

II) ACTION OF DABF-AmB ON THE HIV AND SIV VIRUSES GENERAL PROCEDURES

The N-methylglucamine salt of DABF-AmB is prepared as described inEuropean Application No. 428 440. It is dissolved in a 5% sterilesolution of glucose in water and then diluted in culture medium.

The T cells (Jurkat and CEM) used are very permissive to HIV infection.They are cultured in RPMI 1640 medium supplemented with 10% foetal calfserum, 2 mM of glutamine and a mixture of penicillin and streptomycin.The peripheral blood mononuclear cells are obtained from blood fromhealthy donors, isolated on a Ficoll Hypaque gradient and stimulatedwith 10 μg/ml of phytohaemagglutinin A (PHA), for two days, in RPMI 1640medium supplemented, prior to the infection by HIV, with amikacin andvancomycin (10 mg/ml), polybrene (1 mg/ml) and anti-interferon gammaserum (5 U/ml). After infection, the PBMCs are cultured (10⁵ per well)in flat-bottomed 96-well plates in the same RPMI 1640 medium (200 μl perwell).

Infectious preparations of the LAV/Bru strain of HIV-1 are obtained fromchronically infected CEM cell (clone 13) culture supernatants filteredthrough a 0.45-μm filter.

For the infection, fresh Jurkat or CEM type cells are centrifuged andthe pellet is incubated for 2 hours at 37° C. with the viral preparation(10000 cpm of reverse transcriptase activity/10⁶ cells, whichcorresponds to a multiplicity of infection of 0.04 to 0.1). The cellsare then washed and resuspended (0.5×10^(6/) ml) in fresh culturemedium. Reverse transcriptase activity is determined at specific timesafter the infection, using the acellular infected cell supernatant.

The PBMCs cells (2×10⁶ /ml) are infected with the same viruses and underthe same conditions as the Jurkat or CEM cells. They are then washed andresuspended in fresh culture medium in flat-bottomed 96-well plates (10⁵cells per well in 200 ml).

The reverse transcriptase activity is determined at the indicated timesusing the culture supernatant, according to the procedure described byREY et al., Biochem. Res. Comm. 121, 126-133 (1984).

The viral antigen p24 was quantified in the infected-cell supernatant byradioimmunological assay using the procedure recommended by themanufacturer (ABBOTT, Diagnostics Division).

Membrane expression of CD4 and gp120 is measured by means of themonoclonal antibodies OKT4 (0,5 μg/ml), 110-4 (2 μg/ml) respectively,which are stained using a goat antibody to fluoresceinisothiocyanate-conjugated mouse Ig.

The fluorescence is measured by means of a FACSTAR cell counter (BECTONDICKINSON, MOUNTAIN VIEW, Calif.). The results are expressed as meanfluorescence intensity.

The CD4-specific monoclonal antibody (OKT4 was bought from ORTHOPHARMACEUTICAL CORPORATION (Raritan, N.J.).

The anti-gpl10 monoclonal antibody 110-4 (GENETIC SYSTEMS CORP.,Seattle, Wash.) also recognizes gP120 at the surface of the T cells.

EXAMPLE 1--Action on Reverse Transcriptase Activity

The Jurkat and CEM type T cells are infected with the LAV/BRU strain ofHIV-1.

Viral infection and replication are monitored by measuring the reversetranscriptase activity in the culture supernatants.

As shown in FIG. 3, the reverse transcriptase activity can be detectedfrom the 4th day of the infection for both cell types and a largeactivity peak, at 3.2×10⁶ and 1.5×10⁶ cpm/ml, is observed on the 7th and8th day after the infection, in the Jurkat (FIG. 3a) and CEM (FIG. 3c)cells respectively. The reverse transcriptase activity then decreasesdown to a plateau (about 0.2-0.4×10⁶ cpm/ml). This plateau remainsstable for more than 2 months, and reflects a chronic infection of the Tcells. The infected cells remain viable and within two months followingthe infection, no substantial cell death or inhibition of cell growthare observed.

To measure the anti-HIV activity of DABF-AmB, infected cells arecultured in the presence of increasing doses (between 0.1 μM and 100 μM)of the product; the latter is added immediately after inoculation andthe concentrations are kept constant during the infection by carryingout a one half dilution, every 3 days, of the growing cells in freshmedium containing the desired concentration.

The corresponding results are illustrated in FIGS. 3b and 3d: thereverse transcriptase activity measured on the 8th day of the viralinfection is represented on the y axis and the DABF-AmB concentration onthe x axis.

Inhibition of reverse transcriptase activity is observed both in theJurkat cells (FIG. 3b) and in the CEM cells (FIG. 3d) from 5 μM upwardsand the activity is completely inhibited (inhibition of 88%, N=4) at 10μM of DABF-AmB. The latter concentration was therefore chosen for therest of the studies on the anti-HIV activity of the product. Nosignificant inhibition was observed for DABF-AmB concentrations below 1μM.

Cell viability was estimated at the same time. At concentrations above100 μM, no cytotoxic effect was observed for DABF-AmB either on theJurkat cells or on the CEM cells. It therefore appears that DABF-AmBinhibits the replication of HIV at doses which are not cytotoxic.

It was shown that DABF-AmB has no direct action on reverse transcriptaseactivity and that, consequently, the decrease in this activity cannot bedue to a direct inhibition of the enzyme by DABF-AmB.

EXAMPLE 2--Action on the Expression of the Soluble Viral Antigen p24

The expression of the soluble viral antigen p24 was investigated at thesame time as the reverse transcriptase activity, in the cell culturesupernatants.

The untreated Jurkat cells as well as the untreated CEM cells producelarge amounts of p24 on the 8th day after the infection.

Likewise, as for the reverse transcriptase activity, the continuousaddition of DABF-AmB at a concentration of 10 μM, kept constant for 12days after the infection, substantially decreases the amount of p24antigen (88% inhibition, N=3) in both cell lines.

These results are illustrated in FIG. 4, which represents the productionof antigen p24, 4 days and 8 days after the infection:

▪ Untreated infected cells

DABF-AmB-treated cells

AmB-treated cells (10 μM)

The AmB treatment is carried out under the same conditions as theDABF-AmB treatment. These results show that the efficacy of DABF-AmB onthe multiplication of HIV is substantially greater than that of AmB.

A significant inhibition is observed for DABF-AmB concentrations greaterthan 5 μM.

In another experiment, 10 μM of DABF-AmB are added either duringpretreatment 24 hours before the infection, or alternatively during the2-hour inoculation stage, or alternatively during continuous treatment,as described above, for 12 days following the infection. In this case,the addition of DABF-AmB prior to the infection, or alternatively duringthe 2-hour stage corresponding to viral adsorption, does not bring aboutany significant inhibition of p24 secretion or of reverse transcriptaseactivity.

EXAMPLE 3--Action on the Expression of the Viral Antigen gp120 and theCellular Antigen CD4

The replication of HIV-1 can also be monitored by the expression of theenvelope glycoprotein gp120 at the surface of the infected cells, aswell as by the decrease in the membrane antigen CD4.

Immunofluorescence analyses are carried out on the 8th day of theinfection in order to verify whether the inhibition of HIV replicationby DABF-AmB results in 10 a reduced membrane expression of viralantigens.

On the 8th day of the infection, high levels of gp120 and low levels ofCD4 are observed in the untreated Jurkat and CEM cells (which isverified by the use of the monoclonal antibodies 110.4 and OKT4respectively).

Continuous treatment with 10 μM of DABF-AmB substantially reducesmembrane expression of gp120 in both cell lines. At the same time, thelevel of the membrane antigen CD4 in the DABF-AmB-treated infected cellsincreases until it reaches that for the uninfected cells.

The level of the membrane antigen CD3 is unchanged in the treated oruntreated infected cells.

EXAMPLE 4--Action of DABF-AmB on the Replication of HIV in PBMCs

PBMCs isolated from healthy individuals are infected with HIV-1. Theproduction of antigen p24 as well as the reverse transcriptase activitycan be detected in the supernatants of cultures infected 3 days afterthe infection. The maximum reverse transcriptase activity and p24synthesis are observed between the 6th and 7th day, and the 5th and 6thday respectively. No cell death is observed at this time.

The infected PBMCs are treated continuously as described above for theJurkat and CEM cells, using increasing concentrations of DABF-AmB.

The effect of DABF-AmB on the reverse transcriptase activity and on thesecretion of p24 is evaluated on the 6th day.

As in the case of Jurkat and CEM cells, the product almost completelyinhibits the reverse transcriptase activity as well as the syntheses ofp24 (92±4 and 88±6% inhibition respectively, N=3) at a concentration of10 μM.

Concentrations below 1 μM produce only a low inhibition whereasconcentrations above 20 μM do not bring about a significant increase inthe anti-HIV action.

Moreover, DABF-AmB is without cytotoxic effect on PBMCs atconcentrations ranging up to 10⁻³ M.

It therefore appears that DABF-AmB effectively inhibits the replicationof HIV-1 in normal human T cells at non-cytotoxic concentrations.

EXAMPLE 5--Protection, by DABF-AmB, of CD4 Cells in Cultures ofHIV-infected Human Thymocytes

Human thymuses were obtained during corrective heart surgery operationson 3 to 7 year old children. The tissues were cut into fragments and thered blood cells were removed by treating with Tris-NH₄ Cl₂ buffer. Afterwashing in PBS buffer, cellular suspensions were prepared in an RPMI1640 medium containing 10% of human serum type AD. Dead cells wereremoved by passing over a sterile nylon wool column.

The cells were infected with HIV LAV/BRU as indicated above in thegeneral procedures, and then re-inoculated in the presence of PHA, ofanti-CD2 or anti-CD3 antibodies and of IL2 and cultured for 10 days at arate of 1×10⁷ cells/ml. After 10 days, viral production was evaluated bymeasuring the RT activity in the supernatants and the distribution ofCD4/CD8 was evaluated by double fluorescence flow cytometry by means ofFITC-labelled anti-CD4 antibodies (OKT4) and phycoerythrin-labelledanti-CD8 antibodies (OKT8). DABF-AmB was added to the culturesimmediately after infection and maintained during the entire duration ofthe culture.

DABF-AmB does not significantly modify, even at 10 μM, the distributionof the CD4 and CD8 cells in cultures of uninfected thymocytes activatedwith PHA or with anti-CD2 or anti-CD3 antibodies. A slight increase inthe CD4+ CD8+ population is just about observed.

In the infected and uninfected cultures, a substantial decrease isobserved in the CD4+CD8+ population and in the CD4+CD8- population incorrelation with a high viral production. In the presence of DABF-AmB,the preservation or the reappearance of CD4+ cells in the infectedcultures, in proportion to the concentration of the product. Thisreappearance or this preservation of CD4+ cells are in correlation witha decrease in RT activity in the supernatants.

DABF-AmB therefore inhibits the infection of CD4+ thermocytes by HIV andrestores a normal distribution of the subpopulations of thymic cells inthe infected cultures.

EXAMPLE 6--Action of DABF-AmB on the Early Phases of HIV Infection

HeLa cells transfected with the CD4 gene and capable of being infectedby HIV, carrying the bacterial gene LacZ placed under the control of theLTR of HIV ROCANCOURT et al., J. Virol., 64:2660-2668, (1990)!, wereinfected with HIV-1BRU. 24 hours after the infection of monolayercultures of H12 cells in 1-ml wells, at various viral doses and in thepresence of various concentrations of DABF-AmB, transactivation of theLTR gene by the Tat gene of the virus was revealed by the detection ofblue colonies resulting from the activation of β-galactosidase of theLacZ gene. This detection was carried out after fixing the cells byincubating for one hour in-the presence of5-bromo-4-chloro-3-indoyl-β-D-galactopyranosidase and enzyme substrate.At the concentration of 10 μM, DABF-AmB completely inhibits theappearance of blue colonies, indicating that the product acts early onin the infection cycle.

EXAMPLE 7--Inhibition, by DABF-AmB, of the Cytopathogenic EffectsInduced in HUT78 Cells Infected with the Viruses SIVmac251 and HIV-2-nod

HUT78 cells were inoculated in 96-well plates in an amount of 2×10⁴cells per well, in RPMI 640 medium. They were infected with variousviral concentrations and DABF-AmB was added immediately after theinfection, at various concentrations. The formation of syncytia wasevaluated after 3 and 5 days by direct reading under an invertedmicroscope (semi-quantitative test).

As indicated in Tables II(infection with HIV-2) and III(infection withSIV), DABF-AmB inhibits (-) in a dose-dependent manner and as a functionof the viral content the formation of syncytia in the cultures (+). Itis therefore active on the cytopathogenic effects induced by HIV-2 andby SIV.

                                      TABLE II    __________________________________________________________________________    ANTIVIRAL EFFECTS OF THE COMPOUND DABF-AmB EVALUATED ON    THE CELL LINE HUT78 INFECTED WITH HIV2-rod.                  Controls                          10 TCID50     100 TCID50    DABF-AmB/HUT78/HIV2                  1 2 3 4 5   6   7  8  9   10  11  12    __________________________________________________________________________    neg./pos control                  - - - - ++  +   +  ++ ++  +++ ++  +++    100 μM     - - - - high cytotoxicity                                        -   -   -   -    50 μM      - - - - low cytotoxicity                                        -   -   -   -    10 μM      - - - - -   -   -  -  +   +   -   -     5 μM      - - - - -   +   -  +  +   +   +   +     1 μM      - - - - ++  +   +  ++ +++ ++  ++  +++    0.5 μM     - - - - ++  ++  +  +  +++ ++  ++  +++    0.1 μM     - - - - +   +   ++ +  +++ ++  +++ +++                  Reading after 3 days    __________________________________________________________________________    neg./pos control                  - - - - ++  +   ++ ++ +++ +++ +++ +++    100 μM     - - - - cytotoxicity  -   -   -   -    50 μM      - - - - -   -   -  -  -   -   -   -    10 μM      - - - - -   -   -  -  +   ++  -   -     5 μM      - - - - -   +   -  +  ++  ++  +   +     1 μM      - - - - +++ +++ ++ ++ +++ +++ +++ +++    0.5 μM     - - - - ++  ++  ++ ++ +++ +++ +++ +++    0.1 μM     - - - - +   +   ++ ++ +++ +++ +++ +++                  Reading after 5 days    __________________________________________________________________________

                                      TABLE III    __________________________________________________________________________    ANTIVIRAL EFFECTS OF THE COMPOUND DABF-AmB EVALUATED ON    THE CELL LINE HUT78 INFECTED WITH SIVmac251                  Controls                        10 TCID50                              100 TCID50                                       500 TCID50    DABF-AmB'/HUT78/SIV                  1 2 3 4 5 6 7  8  9  10  11  12    __________________________________________________________________________    neg./pos control                  - - - - - - +  +  +  ++  ++  +    100 μM     - - -   high cytotoxicity                                    -  -   -   -    50 μM      - - - - low cytotoxicity                                    -  -   -   -    10 μM      - - - - - - -  -  -  -   -   -     5 μM      - - - - - - -  -  +  -   +   -     1 μM      - - - - - - +  +  +  ++  +   ++    0.5 μM     - - - - - - +  +  +  +   ++  +    0.1 μM     - - - - - - +  +  +  +   ++  +                  Reading after 3 days    __________________________________________________________________________    neg./pos control                  - - - + + + +  +  +  +++ +++ +++    100 μM     - - - - cytotoxicity                                 -  -  -   -   -    50 μM      - - - 31                          - - -  -  -  -   -   -    10 μM      - - - - - - -  -  +  -   +   +     5 μM      - - - - - + -  -  +  +   +   -     1 μM      - - - + + + +  ++ +  +++ ++  +++    0.5 μM     - - - + + + +  +  ++ ++  +++ ++    0.1 μM     - - - + + + ++ +  +  +   +++ +++                  Reading after 5 days    __________________________________________________________________________

EXAMPLE 8--Inhibition, by DABF-AmB, of the Infection of PBMCs by VariousHIV-1 and by AZT-resistant Viruses

PBMCs were infected under the conditions described above in the generalprocedures, with various HIV-1 isolates derived from patients, oridentified as resistant to AZT. The anti-viral effect of DABF-AmB wasdetermined by measuring the RT activity in the supernatants after 10days of culture.

As shown in FIG. 5, DABF-AmB, at 10 μM, is capable of substantiallyreducing, or even completely inhibiting the infection of PBMC by 10viral strains derived from patients. FIG. 6 shows that this inhibitionis also produced in the case of AZT-resistant strains.

EXAMPLE 9--Inhibition of Cellular Viraemia by DABF-AmB

Lymphocytes from AIDS patients (<200CD4) are isolated on a Ficollgradient using heparinised blood. The cells are taken up in RPMI 1640medium containing antibiotics, 10 U/ml of recombinant IL2 (Boeringher)and anti-interferon alpha serum (anti-IFNα, Sigma) plus 10% of foetalcalf serum. The cells are cultured in 24-well plates in an amount of5×10⁵ cells per well for the first concentration and then subjected to 5fold serial dilutions in the same medium. Half of the medium is replacedtwice weekly. Every week, 3×10⁵ fresh allogeneic PBMCs, derived fromhealthy donors, are added to the cultures. The cultures are kept for 28days in the presence of varying concentrations of DABF-AmB. Vitalproduction is measured weekly by assaying the protein p24 in thesupernatants. Each culture and each assay are carried out in duplicate.

Table IV shows the results obtained for 8 different patients. Thesymbols (-) denote that the amount of p24 detected is at least 95% lessthan the values obtained in the untreated control cultures. Theseresults show that the DABF-AmB molecule inhibits cellular viraemia in 7out of 8 patients. The results obtained in the LOU patient indicateseither a resistance of the virus to the product, or a too high initialviral content. These experiments made it possible to evaluate the IC₅₀for DABF-AmB at between 1 and 5 μM for the different viral isolates(IC50=Concentration of the product inhibiting 50% of the p24 titer inthe culture supernatants, for a patient cells dilution corresponding toa viral content equivalent to 10TCID₅₀.

                                      TABLE IV    __________________________________________________________________________    EFFECT OF DABF-AmB ON CELLULAR VIRAEMIAS                  PHU           VUL            VAC     CAM     SAM           RIF +  +     LOU +   +     MAI +    +       +       +    DAY RIF           MS  PHU                  MS LOU                        MS   VUL                                MS MAI                                      MS   VAC MS  CAM MS  SAM MS    __________________________________________________________________________     0  -  -   -  -  -  -    -  -  -  -    -   -   -   -   -   -     7  +  -   +  -  +  -    +  -  ++ -    14  ++ -   ++ -  ++ ++   ++ -          ++  -   ++  -   +/- -    21  ++ -   ++ -  ++ ++/- ++ -    28  ++ -   ++ -  ++ ++/- ++ -    __________________________________________________________________________

EXAMPLE 10--Action of DABF-AmB on Viral Expression in ReactivatedInfected Cells

The activation of infected CD4+cells is known to stimulate theproduction of the virus. The effect of DABF-AmB was therefore tested onthe vital expression in cells from AIDS patients reactivated in vitro byanti-CD3, by anti-CD28, by PMA (phorbol myristate acetate) or by solubleanti-CD3.

PBMCs from patients (<300 CD4) were isolated on a Ficoll gradient usingblood. The CD4 cells were isolated by cell sorting using a flowcytometer (FACS-Star, Becton-Dickinson, Mountain View, Calif.) afterOKT4-FITC labelling. The CD4 cells were washed again and thenre-inoculated as indicated in 5 and activated either with anti-CD3 oranti-CD28 antibodies or with PMA. Viral expression was evaluated after10 days by ELISA assay of p24 in the supernatants.

The results presented in Table 4 show that in 7 patients tested,DABF-AmB inhibits viral expression by more than 90% at a concentrationof 10 μM. These results show that DABF-AmB is capable of inhibitingvirus production during reactivation of preinfected cells.

                                      TABLE V    __________________________________________________________________________    STUDY OF HIV PRODUCTION IN VITRO REACTIVATED CD4    PERIPHERAL BLOOD T CELLS FROM SEROPOSITIVE PATIENTS           STIMULATION                                  cCD3 +               cCD3 +   CD28 +                             cCD3 °                                  CD28 +   PMA +    sCD3 +    PATIENTS           cCD3               MS   CD28                        MS   CD28 MS   PMA MS   sCD3                                                    MS    __________________________________________________________________________    S23 (PP)                           +   -    S24 (I)                            +/- -    S25 (PP)    S26 (PP)           +/-               ++   -    S27 (PP)    S28 (PP)    S29 (I)                  +    -    S30 (I)           +/- -    ++       ++   -    S31 (PP)        +   -    S32 (PP)           +   -    ++  -    +    -             +   +/-    S33 (I)    S34 (PP)    S35    S36    __________________________________________________________________________

II)--ACTION OF DABF-AmB ON THE RHINOVIRUS HRV14 EXAMPLE 11--Inhibition,by DABF-AmB, of the cytopathogenic effects of HRV14

HeLa-Ohio cells cultured until they have become confluent, are infectedwith a suspension of HRV14 virus. The cytopathogenic effect of the virusis evaluated by staining the cells with a 0.5% solution of crystalviolet and 20% of methanol; after rinsing the plates with water, theoptical density is read at 550 nm.

For the rest of the experiment, the cells are infected with a viralsuspension with a titre such that 50% of the cells are killed after 3days on control cells not receiving any treatment. 1 hour after theinfection, the test product (AmB or DABF-AmB) is added at concentrationsranging from 0.03 to 100 μM. The percentage inhibition of thecytopathogenic effect is determined as follows:

     OD (treated infected cells)-OD (infected cells)!

     OD (healthy cells)-OD (infected cells)!

Under these conditions, AmB has no detectable antiviral activity and iscytotoxic at concentrations above 5 μM. In contrast, DABF-AmB completelyinhibits the cytopathogenic effects of the virus from 10 μM upwards IC₅₀(50% inhibition of the cytopathogenic effect)=5 μM!, and no cytotoxiceffect was observed for DABF-AmB after treating the cells with 20 μM for4 weeks. N-methylglucamine and benzylidene glucoside have no effect onthe virus.

When the suspension of HRV14 is preincubated at 37° C. in the presenceof DABF-AmB for 5 hours, no inhibition of the cytopathogenic effect isobserved compared with a control suspension incubated under the sameconditions in the absence of DABF-AmB. DABF-AmB therefore has no directvirucidal effect on HRV14.

The effect of the addition of DABF-AmB was tested at various times:preincubation of the cells with DABF-AmB before the viral infection;addition of DABF-AmB at the time of the infection; addition of DABF-AmBafter the infection, during the period of viral proliferation. Theresults obtained show that DABF-AmB acts essentially during viralproliferation.

As evident from the above, the invention is not in the least limited tothose of its embodiments and applications which have just been describedmore explicitly; on the contrary, it embraces all the variants which maycome to the mind of a specialist in this field without departing fromthe framework or the scope of the present invention.

I claim:
 1. A method for inhibiting the multiplication cycle of a vitalagent in host cells wherein said multiplication cycle entails a serineprotease comprising:contacting said viral agent and host cells with aneffective serine protease inhibiting amount of a compound of formula (I)##STR3## where R₁ represents a polyene macrolide of amphotericin B, R₂represents a hydrogen atom or a methyl group, and R₃ represents amethyl, ethyl, propyl or phenyl group.
 2. The method of claim 1, whereinthe compound of formula (I) is1-deoxy-1-amino-4,6-O-benzylidene-D-fructosyl-AmB.
 3. The method ofclaim 1, wherein said compound is used at a concentration from 1 mM to100 mM.